Modified PSMA ligands and uses related thereto

ABSTRACT

The instant invention provides reagents and methods for diagnosis, detection and treatment of cancers (for example, prostate cancers). In particular, the invention provides methods to generate various functionalized PSMA ligands, and their uses in diagnosis, detection, imaging, and treatment of prostate cancers, especially those overexpressing PSMA.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional application60/267,055, filed on Feb. 7, 2001, the specifications of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Prostate cancer is the second leading cause of cancer-related deaths inUS men. It is estimated that 184,500 new cases of prostate cancer willbe diagnosed in the United States in 1998, and over 39,200 deaths willresult from this cancer.

Prostate-specific membrane antigen (PSMA) is a 120 kDa protein expressedin prostate tissues and was originally identified by reactivity with amonoclonal antibody designated 7E11-C5 (Horoszewicz et al., 1987,Anticancer Res. 7:927-935; U.S. Pat. No. 5,162,504). PSMA ischaracterized as a type II transmembrane protein sharing sequenceidentity with the transferrin receptor (Israeli et al., 1994, CancerRes. 54:1807-1811). PSMA is a glutamate carboxy-peptidase that cleavesterminal carboxy glutamates from both the neuronal dipeptideN-acetylaspartylglutamate (NAAG) and gamma-linked folate polyglutamate.That is, expression of PSMA cDNA confers the activity of N-acetylatedα-linked acidic dipeptidase or “NAALADase” activity (Carter et al.,1996, PNAS 93:749-753).

More importantly, PSMA is expressed in increased amounts in prostatecancer, and elevated levels of PSMA are also detectable in the sera ofthese patients (Horoszewicz et al., 1987, supra; Rochon et al., 1994,Prostate 25:219-223; Murphy et al., 1995, Prostate 26:164-168; andMurphy et al., 1995, Anticancer Res. 15:1473-1479). As a prostatecarcinoma marker, PSMA is believed to serve as a target for imaging andcytotoxic treatment modalities for prostate cancer. Prostatecarcinogenesis, for example, is associated with an elevation in PSMAabundance and enzymatic activity of PSMA. PSMA antibodies, particularlyindium-111 labeled and tritium labeled PSMA antibodies, have beendescribed and examined clinically for the diagnosis and treatment ofprostate cancer. PSMA is expressed in prostatic ductal epithelium and ispresent in seminal plasma, prostatic fluid and urine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Guilford 11254-36 was conjugated under aqueous conditions tothe near-infrared fluorophore IRDye78 (LI-COR, Lincoln, Nebr.;excitation 771 nm, emission 796 nm) using the chemistry shown.

FIG. 1B. The desired product of the reaction could be separated easilyfrom reactants using normal phase thin-layer chromatography.

FIG. 2. COS-7 cells, which normally do not express PSMA, were infectedby adenovirus constructs expressing GFP, or co-expressing GFP and PSMA.Top panel shows near-infrared fluorescence (excitation 771 nm, emission796 nm). Middle panel shows green fluorescent protein signal. Bottompanel shows phase contrast of same field. Arrow points to representativecell with nuclear/cytoplasmic GFP signal and strong plasma membraneGuilford/IRDye78 signal. Control cells expressing GFP only (rightcolumn) do not bind Guilford/IRD78 conjugate.

FIG. 3. COS-7 cells, which normally do not express PSMA, were infectedby adenovirus constructs co-expressing GFP and cell surface proteinErb-b2, or co-expressing GFP and PSMA. Top panel shows near-infraredfluorescence (excitation 771 nm, emission 796 nm). Middle panel showsgreen fluorescent protein signal. Bottom panel shows phase contrast ofsame field. Arrow points to representative cell with strong plasmamembrane Guilford/IRDye78 signal in cells making PSMA. No binding isseen with cells expressing Erb-B2 (right panel).

FIG. 4. PC-3 cells, a human prostate cancer cell line which normally donot express PSMA, were infected by adenovirus constructs co-expressingGFP and cell surface protein Erb-b2, or co-expressing GFP and PSMA. Themiddle column represents uninfected control PC-3 cells. The first rowshows near-infrared fluorescence (excitation 771 nm, emission 796 nm).The second row shows immunostaining using either Erb-b2 or PSMA specificantibodies. The third row shows green fluorescent protein signal. Thelast row shows phase contrast of same field. Strong plasma membraneGuilford/IRDye78 signal is seen only in cells making PSMA. No binding isseen with cells expressing Erb-B2 (right panel) or control PC-3 cells.

FIG. 5. Top panel: β-Asp-Glu (β-AG) is an inhibitor of PSMA enzymaticactivity as measured by a ³H-NAAG assay. The vertical axis representswild-type PSMA activity in percentage (100% represents full wild-typeactivity). The inhibitory activity of β-AG is compared to a PSMAcompetitive inhibitor beta-N-acetyl-AG (β-NAAG). Interestingly, IRDye78is itself found to be a potent inhibitor of PSMA, and the conjugatedβ-AG/IRDye78 (β-AG/78) exhibits a synergistic inhibitory effect to PSMAwhen compared to either β-AG or unconjugated IRDye78. Bottom panel:structure of the conjugated β-AG/IRDye78 (β-AG/78).

FIG. 6. Top panel: purification of β-AG/IRDye78 (β-AG/78) wasaccomplished by either TLC (not shown) or HPLC. Bottom panel: structureof β-AG.

FIG. 7. Top panel: the Guilford/IRDye78 conjugate has a potency for PSMAinhibition that is synergistic with either compound alone. PSMAenzymatic activity is measured by a ³H-NAAG assay. The vertical axisrepresents wild-type PSMA activity in percentage (100% represents fullwild-type activity). The inhibitory activity of Guilford/IRDye78 iscompared to IRDye78 and Guilford 11254-36, the structure of which isshown in the lower panel.

FIG. 8. The conjugates (beta-AG/78 and Guilford/78) created byconjugation to the primary amine not only inhibit activity, but can alsobe proven to bind directly to the PSMA molecule. As shown in thefluorescence polarization data, both beta-AG/78 and Guilfrod/78 bindsdirectly to PSMA with a K_(d) essentially equivalent to their respectiveIC₅₀ of enzyme inhibition. This means that IC₅₀ can be used as asurrogate for affinity, and also predicts that the conjugates presentedherein can be used as visualization agents for PSMA-positive cells.

FIG. 9. Structural comparison of a few known inhibitors of PSMA.

FIG. 10. Computer-aided 3-D molecular modeling of PSMA ligands. The 3-Dmolecular modeling was performed using MM2 energy minimization (CSChem3D Pro software) in the presence of the Zn²⁺ atom known to be at thecatalytic site of PSMA. By comparing the structures of PSMA naturalsubstrate L-Asp-L-Glu to various inhibitors, it was revealed that thespacing of carboxylic acids and negative charge plays important role indetermining binding affinity. The presence of the N-acetyl group or acomplete Asp molecule is not required for binding.

FIG. 11. Two exemplary modified PSMA ligands. Top panel shows aradioscintigraphic probe for prostate cancer detection comprising aradiometal chelator coupled to the primary amine of Guilford 11254-36.Bottom panel shows a cytotoxic drug for prostate cancer treatmentcomprising doxorubicin coupled to Guilford 11254-36.

SUMMARY OF THE INVENTION

One aspect of the invention provides a compound represented in thegeneral formula (Ia):

-   -   wherein:    -   X represents O or S;    -   Y represents:    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R1 and R3, independently for each occurrence, represents an        alkyl, an alkenyl, a cycloalkyl, a cycloalkenyl, an aryl,        —(CH₂)_(m)-aryl, -alkyl-CO₂R4, -alkenyl-CO₂R4,        -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or -aryl-CO₂R4;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   D₁ represents O or S;    -   D₂ represents N₃, SH₂, NH₂, or NO₂;    -   m is 1, 2, 3 or 4; and,    -   n is 0, 1, 2 or 3.

A related aspect provides a compound represented in the general formula(Ib):

-   -   wherein X, Y, R, R1-R4, D1, D2, m and n are defined as above.

Another related aspect provides a compound represented in the generalformula (Id):

-   -   wherein X, Y, R, R1-R4, D1, D2, m and n are defined as above.

Another related aspect provides a compound represented in the generalformula (Ie):

-   -   wherein X, Y, R, R1-R4, D1, D2, m and n are defined as above.

Another related aspect provides a compound represented in the generalformula (If):

-   -   wherein X, Y, R, R1-R4, D1, D2, m and n are defined as above.

In a preferred embodiment, X represents O. In another preferredembodiment, R1 and R3 each independently represent a -lower alkyl-CO₂R4.In another preferred embodiment, Y represents —P(═O)(—OR2)—. In anotherpreferred embodiment, R2 represents H or a lower alkyl. Most preferably,R2 represents H.

Another related aspect provides a compound represented in the generalformula (Ic):

-   -   wherein X, Y, R, R1-R4, D1, D2, m and n are defined as above.

In a preferred embodiment, X represents O. In another preferredembodiment, R1 represent H, a -lower alkyl-CO₂R4, or —(CH₂)_(m)-aryl. Inanother preferred embodiment, Y represents —P(═O)(—OR2)—. In anotherpreferred embodiment, R2 represents H or a lower alkyl. Most preferably,R2 represents H.

Another related aspect provides a compound represented in the generalformula (II):

-   -   wherein:    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R3 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, —(CH₂)_(m)-aryl, -alkyl-CO₂R4,        -alkenyl-CO₂R4, -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or        -aryl-CO₂R4;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   m is 1, 2, 3 or 4; and,    -   n is 0, 1, 2 or 3.

Another related aspect provides a compound represented in the generalformula (III):

-   -   wherein:    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R3 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, —(CH₂)_(m)-aryl, -alkyl-CO₂R4,        -alkenyl-CO₂R4, -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or        -aryl-CO₂R4; R2 and R4, independently for each occurrence,        represent hydrogen, a lower alkyl, or a pharmaceutically        acceptable salt;    -   R5 represents H or a lower alkyl;    -   m is 1, 2, 3 or 4; and    -   n is 0, 1, 2 or 3.

Another related aspect provides a compound represented in the generalformula (IV):

-   -   wherein R, R2-R5, m and n are defined above.

Another related aspect provides a compound represented in the generalformula (V):

-   -   wherein    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   R6 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, or —(CH₂)_(m)-aryl; and,    -   m is 1, 2, 3 or 4.

In a preferred embodiment, R is at least 25 amu in size, morepreferably, at least 50 amu, at least 100 amu in size, or at least 250amu in size.

In another preferred embodiment, R is a cytotoxic moiety, and R ishydrolyzable from the PSMA ligand.

In another preferred embodiment, R is linked to the rest of the moleculeby use of an amide or ester group. For example, R can be linked to therest of the molecule by use of an acid labile or base-cleavable linker.

In another preferred embodiment, R is a chelate moiety for chelating ametal. For example, R can be a chelator for a radiometal or aparamagnetic ion. Specifically, R can be a chelator for a radionuclideuseful for radiotherapy or imaging procedures. In a preferredembodiment, R is a beta- or alpha-emitter for radio-therapeutic use. Inanother preferred embodiment, R can be a gamma-emitter,positron-emitter, Auger electron-emitter, X-ray emitter orfluorescence-emitter. In a most preferred embodiment, R is ^(99m)Tc(technium).

In another preferred embodiment, R is a radiosensitizing agent selectedfrom: nitroimidazoles, metronidazole or misonidazole.

In another preferred embodiment, R is a bifunctional chelator N_(x)S_(y)that are capable of coordinately binding a metal or radiometal, whereinx and y are integers between 1 and 4. N_(x)S_(y) can have a N₂S₂ or aN₃S core.

In another preferred embodiment, R is Boron addend.

In another preferred embodiment, R is a chemotherapeutic agent.

In another preferred embodiment, R is a drug that interferes withintracellular protein synthesis.

In another preferred embodiment, R is a prodrug that is only activatedfrom its inactive precursor form by host metabolism.

In another preferred embodiment, R is a toxin selected from: ricin,ricin A chain (ricin toxin), Pseudomonas exotoxin (PE), diphtheria toxin(DT), Clostridium perfringens phospholipase C (PLC), bovine pancreaticribonuclease (BPR), pokeweed antiviral protein (PAP), abrin, abrin Achain (abrin toxin), cobra venom factor (CVF), gelonin (GEL), saporin(SAP), modeccin, viscumin or volkensin.

In another preferred embodiment, R is an enzyme that converts prodruglocally.

Another aspect of the invention provides a pharmaceutical compositioncomprising the compound of any one of the modified PSMA ligands of theinstant invention, and a pharmaceutically acceptable carrier.

Another aspect of the invention provides a method for detecting orimaging PSMA (prostate-specific membrane antigen)-expressing cells in apatient, comprising:

-   -   (a) contacting the patient with a modified PSMA ligand of any        one of claims 1-5, 11, and 17-20;    -   (b) detecting the modified PSMA ligand, thereby detecting        PSMA-expressing cells in the patient.

In a preferred embodiment, the PSMA-expressing cells are prostatic cellsin prostatic hyperplasia or prostate cancer. In another preferredembodiment, the modified PSMA ligand is modified by an imaging agent.For example, the imaging agent is a radionuclide imaging agent. Theradionuclide imaging agent can be radioactive iodine or indium.

In another preferred embodiment, the modified PSMA ligand is detected byradioscintigraphy, magnetic resonance imaging (MRI), computed tomography(CT scan), or positron emission tomography (PET).

In another preferred embodiment, the contacting step (a) is effected byadministering to the patient the modified PSMA ligand. In anotherpreferred embodiment, the detecting step (b) includes determining thevolume, shape and/or location of PSMA-expressing cells in the patient.

Another aspect of the invention provides a method for determining theabundance of PSMA in a sample, comprising:

-   -   (a) contacting the sample with any one of the modified PSMA        ligands of claims 1-5, 11, and 17-20;    -   (b) determining the abundance of the modified PSMA ligands bound        to PSMA, or the abundance of the modifying group of said bound        ligands, thereby determining the abundance of PSMA in said        sample.

In a preferred embodiment, the sample is prostatic fluid, urine, orobtained from seminal plasma.

Another aspect of the invention provides a method to diagnose, in a testsample, the presence of a prostate disease condition associated withPSMA-overexpression, comprising:

-   -   (a) using the method of claim 52, determining the abundance of        PSMA in the test sample and a normal control sample;    -   (b) comparing the level of abundance of PSMA in the test sample        and the control sample;    -   wherein statistically significant higher levels of abundance of        PSMA in the test sample indicates the presence of a prostate        disease condition associated with PSMA-overexpression.

Another aspect of the invention provides a method to treat a patientsuffering from a disease condition associated with PSMA-overexpression,comprising administering to the patient an effective amount of modifiedPSMA ligand of the instant invention.

In a preferred embodiment, the disease condition is prostatichyperplasia or prostate cancer. In another preferred embodiment, themodified PSMA ligand is modified by a cytotoxic agent. In anotherpreferred embodiment, the modified PSMA ligand is modified by aradiometal chelating agent. In a preferred embodiment, the methodfurther comprises infusing into the patient an effective amount ofchelator compounds, which can be EDTA or DTPA. In a preferredembodiment, the modified PSMA ligand is administered to the patient at adose that contain 10-100 times less active agent as an active moietythan the dosage of agent administered as unconjugated active agents.

Another aspect of the invention provides a kit for diagnosing ordetecting the presence of a PSMA, comprising: a) at least one of themodified PSMA ligand of any one of claims 1-5, 11, and 17-20; b) aninstruction.

In a preferred embodiment, the modified PSMA ligand contains a chelatemoiety for chelating a metal or a paramagnetic ion. In another preferredembodiment, the kit further comprises at least one metal. For example,the metal can be a radionuclide useful for radiotherapy or imagineprocedures.

Another aspect of the invention provides a method to treat a patientsuffering from a disease condition associated with PSMA-overexpression,comprising administering to the patient an effective amount of IRDye78.

DETAILED DESCRIPTION OF THE INVENTION

(i) Overview

The present invention is directed to improved reagents for detectingcells based on NAALADase activity, PSMA binding, and/or for selectivekilling of cells in a NAALADase-dependent manner. The invention derivesin part from the discovery that relatively large functionalities can beadded to modified PSMA ligands without disrupting the binding of thoseinhibitors to the enzyme. Accordingly, modified PSMA ligands can bederivitized with, and used to selectively deliver, such secondaryfunctionalities as cytotoxic agents, radiometal chelating agents,fluoremetric agents and other imaging agents. For ease of reading, thesecompounds are referred to herein as the subject “modified (orfunctionalized) PSMA ligands.”

One aspect of the present invention provides a method for detecting oridentifying cells which express a NAALADase activity, e.g., PSMA, andcan be used for example to detect the presence of prostatic hyperplasiaor prostate cancer. According to the present invention, the modifiedPSMA ligand may be an imaging agent. Imaging agents are usefuldiagnostic procedures as well as the procedures used to identify thelocation of metastasized cells. For instance, imaging using the subjectmodified PSMA ligands, e.g., those including an imaging agentfunctionality, can be performed by radioscintigraphy, magnetic resonanceimaging (MRI) or computed tomography (CT scan). The most commonlyemployed radionuclide imaging agents include radioactive iodine andindium. Imaging by CT scan may employ a heavy metal such as ironchelates. MRI scanning may employ chelates of gadolinium or manganese.Additionally, positron emission tomography (PET) may be possible usingpositron emitters of oxygen, nitrogen, iron, carbon, or gallium. Forinstance, the subject modified PSMA ligands, e.g., those including aimaging agent functionality, can be administered to a patient and usedas part of a detection protocol to image tissue in a manner dependent onthe level of expression of PSMA. In such a manner, the volume, shape andlocation of hyperproliferative prostate tissue can be imagined in thebody.

In a related assay, forms of the modified PSMA ligand which are amenableto detection by, e.g., spectrometric techniques or scintillation, can beused to determine the level of PSMA in samples of prostate tissue orbodily fluid, and compared to reference samples, in to ascertain thePSMA levels/NAALADase activity relative to normal prostate tissue.

Another aspect of the invention utilizes forms of the modified PSMAligands to selectively ablate tissue having elevated levels of PSMA,e.g., as part of a therapeutic regimen to lessen the severity ofprostatic hyperplasia or prostate cancer. In such embodiments, versionsof the modified PSMA ligand which include such secondary functionalitiesas cytotoxic agents or radiometal chelating agents can be used.

Still another aspect of the invention provides compositions and kitsincluding the subject modified PSMA ligands.

(ii) Definitions

Before describing exemplary embodiments, there is provided certaindefinitions for terms used in the specification and claims.

“NAAG” refers to N-acetyl-aspartyl-glutamate, an important peptidecomponent of the brain, with levels comparable to the major inhibitorneurotransmitter gamma-aminobutyric acid (GABA). NAAG isneuron-specific, present in synaptic vesicles and released upon neuronalstimulation in several systems presumed to be glutamatergic. Studiessuggest that NAAG may function as a neurotransmitter and/orneuromodulator in the central nervous system, or as a precursor of theneurotransmitter glutamate. “Beta-NAAG” or “β-NAAG” is a moleculesimilar to the natural substrate NAAG. β-NAAG replaces the naturalAsp-Glu linkage with a linkage of Glu to the beta-carbon of Asp. β-NAAGis a non-hydrolizable competitive inhibitor of PSMA. “Beta-AG” or “β-AG”is essentially the same as beta-NAAG except that it lacks the N-acetylmoiety (see FIG. 6, lower panel). Beta-NAAG can not be hydrolyzed byPSMA.

“NAALADase” refers to N-acetylated α-linked acidic dipeptidase, amembrane-bound metallopeptidase which catabolizes NAAG toN-acetylaspartate (NAA) and glutamate (GLU):

NAALADase shows a high affinity for NAAG with a K_(m) of 540 nM. If NAAGis a bioactive peptide, then NAALADase may serve to inactivate NAAG'ssynaptic action. Alternatively, if NAAG functions as a precursor forglutamate, the primary function of NAALADase may be to regulate synapticglutamate availability.

The term “prevention,” in relation to tumor growth or tumor cell growth,means no tumor or tumor cell growth if none had occurred, no furthertumor or tumor cell growth if there had already been growth.

The term “prostate disease” relates to prostate cancer such asadenocarcinoma or metastatic cancers, conditions characterized byabnormal growth of prostatic epithelial cells such as benign prostatichyperplasia, and other conditions requiring treatment by the compoundsof the present invention.

“PSA” refers to Prostate Specific Antigen, a well known prostate cancermarker. It is a protein produced by prostate cells and is frequentlypresent at elevated levels in the blood of men with prostate cancer. PSAcorrelates with tumor burden, serves as an indicator of metastaticinvolvement, and provides a parameter for following a prostate cancerpatient's response to surgery, irradiation and androgen replacementtherapy.

“PSMA” refers to Prostate Specific Membrane Antigen, a potentialprostate carcinoma marker that has been hypothesized to serve as atarget for imaging and cytotoxic treatment modalities for prostatecancer. PSMA is expressed in prostatic ductal epithelium and is presentin seminal plasma, prostatic fluid and urine. It has been found that theexpression of PSMA cDNA confers the activity of NAALADase.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein an animal, including a human being, issubject to medical aid with the object of improving the animal'scondition, directly or indirectly.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m is zero or an integer in the range of 1 to 6, and R₈ representsa substituted or unsubstituted aryl, an aralkyl, a cycloalkyl, acycloalkenyl, or a heterocycle.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

-   -   wherein R₉, R₁₀ and R′₁₀ each independently represent a        hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀        taken together with the N atom to which they are attached        complete a heterocycle having from 4 to 8 atoms in the ring        structure; R₈ represents an aryl, a cycloalkyl, a cycloalkenyl,        a heterocycle or a polycycle; and m is zero or an integer in the        range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀        can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do        not form an imide. In even more preferred embodiments, R₉ and        R₁₀ (and optionally R′₁₀) each independently represent a        hydrogen, an alkyl, an alkenyl, or —(CH₂)_(m)—R₈. Thus, the term        “alkylamine” as used herein means an amine group, as defined        above, having a substituted or unsubstituted alkyl attached        thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from heteroatoms(preferably 1 to 4), for example, benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

-   -   wherein X is a bond or represents an oxygen or a sulfur, and R₁₁        represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(n)—R₈ or a        pharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an        alkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as        defined above. Where X is an oxygen and R₁₁ or R′₁₁ is not        hydrogen, the formula represents an “ester.” Where X is an        oxygen, and R₁₁ is as defined above, the moiety is referred to        herein as a carboxyl group, and particularly when R₁₁ is a        hydrogen, the formula represents a “carboxylic acid.” Where X is        an oxygen, and R′₁₁ is hydrogen, the formula represents a        “formate.” In general, where the oxygen atom of the above        formula is replaced by sulfur, the formula represents a        “thiocarbonyl” group. Where X is a sulfur and R′₁₁ or R′₁₁ is        not hydrogen, the formula represents a “thioester.” Where X is a        sulfur and R₁₁ is hydrogen, the formula represents a        “thiocarboxylic acid.” Where X is a sulfur and R′₁₁ is hydrogen,        the formula represents a “thiolformate.” On the other hand,        where X is a bond, and R₁₁ is not hydrogen, the above formula        represents a “ketone” group. Where X is a bond, and R₁₁ is        hydrogen, the above formula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂ ⁻.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings.” Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

The term “modifying group” or “functional group” refers to thefunctional “R groups” of the modified PSMA ligands. It could be afluorescent tag, a chelate ligand, a cytotoxic moiety, or any otherfunctional groups.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to bind to PSMA), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound. In general, the compoundsof the present invention may be prepared by the methods illustrated inthe general reaction schemes as, for example, described below, or bymodifications thereof, using readily available starting materials,reagents and conventional synthesis procedures. In these reactions, itis also possible to make use of variants which are in themselves known,but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67^(th) Ed., 1986-87, inside cover.Also for purposes of this invention, the term “hydrocarbon” iscontemplated to include all permissible compounds having at least onehydrogen and one carbon atom. In a broad aspect, the permissiblehydrocarbons include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic organic compoundswhich can be substituted or unsubstituted.

(iii) Exemplary Modified PSMA Ligands

The natural substrate for PSMA is the dipeptide N-Acetyl-L-Asp-L-Glu(NAAG). The first competitive inhibitor described for PSMA (Serval etal., J. Neurochemistry, 1990, 55: 39-46) is beta-NAAG, a moleculesimilar to the natural substrate NAAG. Beta-NAAG replaces the naturalAsp-Glu linkage with a linkage of Glu to the beta-carbon of Asp (seemodeling below). Since the N-acetyl moiety of NAAG is reportedly notessential for NAALADase specificity (Serval et al., J. Neurochemistry,1990, 55: 39-46), it is our prediction that the N-terminus of NAAG orbeta-NAAG may not participate directly in binding the enzyme, and thusmodifications of the N-terminus of NAAG, beta-NAAG, beta-AG, or otherPSMA ligands by extension with functional groups (e.g. fluorophore,radiometal chelators, cytotoxic agents, etc.) may not dramaticallyaffect the binding affivnity to PSMA.

In fact, a direct comparison of the chemical structures of severalpotential PSMA ligands, namely NAAG, beta-NAAG, a non-competitiveinhibitor called quisqualic acid, and a high affinity compoundsynthesized by Zeneca and Guilford pharmaceuticals called 2-PMPA(molecule 3 in Jackson et al., J. Med. Chem. 1996, 39: 619-622), yieldsvaluable information regarding the structural characteristics of PSMAbinding region (FIG. 9).

While all depicted compounds and their derivatives are PSMA ligands withvarious degrees of inhibitory functions, it is contemplated that in apreferred embodiment of the invention, there is a precise spacing ofcarboxylic acid residues on the right hand side of PSMA ligands, and alocalization of negative charge at the left hand side of each molecule.In another preferred embodiment of the invention, the region to the leftof the PSMA binding site (for example, the left of the phosphate groupof the Guilford compound 3) can be further extended to accommodateaddition of modification groups, without significant loss ofaffinity/inhibitory activity for PSMA (FIG. 9).

There are many conceivable ways to modify these PSMA ligands. Forexample, in a preferred embodiment, an extended region can be added tothe left of the Guilford compound 3, which further includes at least one—NH2 group for conjugation of modification groups (see below).

Careful analysis reveals that this compound is actually an amino acidwith an unnatural R-group, and chemical synthesis may be simplifiedusing this perspective. Also, such a molecule is chiral with respect tothe “alpha” carbon, and hence two possible enantiomers can both betested.

Alternatively, in another preferred embodiment, the amino (or othercoupling group) could be coupled to the first methylene group (seebelow):

In another preferred embodiment, a modification group can be added tothe —NH2 group in β-AG or its derivative, while in another preferredembodiment, a modification group can be added to the —NH— group in AG orits derivative.

Maintenance of binding affinity by these modifications can be routinelydetermined empirically. Computer modeling may also be used to aid thedesign of certain modifications, as exemplified in FIG. 10. For both ofthese preferred compounds, functional groups can be added to the freeamino group by a single step coupling using NHS-esters or EDC activatedcarboxyl groups, thus generating functionalized or modified PSMA ligandsuseful for cancer detection and/or treatment. Additional spacing atomsbetween the amino group of the above two compounds and the functionalgroup can be routinely determined empirically.

For example, FIG. 11 shows two exemplary modified PSMA ligands thatutilizes the primary amine of one of the listed preferred compounds toconjugate functional groups.

In one embodiment, the subject modified PSMA ligand is represented inthe general formula (Ia):

Wherein:

-   -   X represents O or S;    -   Y represents:    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R1 and R3, independently for each occurrence, represents an        alkyl, an alkenyl, a cycloalkyl, a cycloalkenyl, an aryl,        —(CH₂)_(m)-aryl, -alkyl-CO₂R4, -alkenyl-CO₂R4,        -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or -aryl-CO₂R4;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   D₁ represents O or S;    -   D₂ represents N₃, SH₂, NH₂, or NO₂;    -   m is 1, 2, 3 or 4; and,    -   n is 0, 1, 2 or 3.

In other embodiments, the subject modified PSMA ligand is represented inthe general formula (Ib):

-   -   wherein X, Y, R, R1, R3 and R4 are as defined above.

In other embodiments, the subject modified PSMA ligand is represented inthe general formula (Ic):

-   -   wherein X, Y, R, R1, R3 and R4 are as defined above.

In other embodiments, the subject modified PSMA ligand is represented inthe general formula (Id):

-   -   wherein X, Y, R, R1, R3 and R4 are as defined above.

In other embodiments, the subject modified PSMA ligand is represented inthe general formula (Ie):

-   -   wherein X, Y, R, R1, R3 and R4 are as defined above.

In other embodiments, the subject modified PSMA ligand is represented inthe general formula (If):

-   -   wherein X, Y, R, R1, R3 and R4 are as defined above.

In certain preferred embodiments of compounds Ia, Ib, Id-If: Xrepresents O; R¹ and R³ each independently represent a -loweralkyl-CO₂R⁴; Y represents —P(═O)(—OR2)—; and R² represents H or a loweralkyl, and more preferably H.

In certain preferred embodiments of compound Ic: X represents O; R¹represent H or a -lower alkyl-CO₂R⁴; —(CH₂)_(m)-aryl; Y represents—P(═O)(—OR2)—; and R² represents H or a lower alkyl, and more preferablyH.

Unless apparent from the context, references to “formula I” throughoutthe application mean any one of formulas Ia-If.

For example, the subject modified PSMA ligand may be represented in thegeneral formula (II):

-   -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R3 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, —(CH₂)_(m)-aryl, -alkyl-CO₂R4,        -alkenyl-CO₂R4, -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or        -aryl-CO₂R4;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   m is 1, 2, 3 or 4; and,    -   n is 0, 1, 2 or 3.

In yet other embodiments, the chelate ligand, fluorescence tag or acytotoxic moiety can be covalently linked via one of the carboxylicgroups, e.g., as represented in Formula III, or IV:

Wherein:

-   -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R3 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, —(CH₂)_(m)-aryl, -alkyl-CO₂R4,        -alkenyl-CO₂R4, -cycloalkyl-CO₂R4, -cycloalkenyl-CO₂R4 or        -aryl-CO₂R4;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   R5 represents H or a lower alkyl;    -   m is 1, 2, 3 or 4; and    -   n is 0, 1, 2 or 3.

In still other embodiments, the subject modified PSMA ligand isrepresented in Formula V:

-   -   wherein    -   R represents a chelate ligand, a fluorescence tag, or a        cytotoxic moiety;    -   R2 and R4, independently for each occurrence, represent        hydrogen, a lower alkyl, or a pharmaceutically acceptable salt;    -   R6 represents an alkyl, an alkenyl, a cycloalkyl, a        cycloalkenyl, an aryl, or —(CH₂)_(m)-aryl; and,    -   m is 1, 2, 3 or 4.

In many embodiments of the subject ligands, the secondary functionalityR will be relatively large, e.g., at least 25 amu in size, and in manyinstances can be at least 50, 100 or 250 amu in size.

In certain preferred embodiments, particularly where R is a cytotoxicmoiety, R is hydrolyzable from the PSMA ligand, e.g., such as may beprovided by use of an amide or ester group linking R to the rest of themolecule.

In certain preferred embodiments, R is a chelate moiety for chelating ametal, e.g., a chelator for a radiometal or paramagnetic ion. Inpreferred embodiments, R is a chelator for a radionuclide useful forradiotherapy or imaging procedures. Radionuclides useful within thepresent invention include gamma-emitters, positron-emitters, Augerelectron-emitters, X-ray emitters and fluorescence-emitters, with beta-or alpha-emitters preferred for therapeutic use. Examples ofradionuclides useful as toxins in radiation therapy include: ³²P, ³³P,⁴³K, ⁴⁷Sc, ⁵²Fe, ⁵⁷Co, ⁶⁴Cu, ⁶⁷Ga, ⁶⁷Cu, ⁶⁸Ga, ⁷¹Ge, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br,⁷⁷As, ⁷⁷Br, ⁸¹Rb/^(81M)Kr, ^(87M)Sr, ⁹⁰y, ⁹⁷Ru, ⁹⁹Tc, ¹⁰⁰Pd, ¹⁰¹Rh, ¹⁰³Pb, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹¹⁹Sb ¹²¹Sn, ¹²³I, ¹²⁵I, ¹²⁷Cs¹²⁸Ba, ¹²⁹Cs, ¹³¹I, ¹³¹Cs, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹³⁶Ho, ¹⁶⁹Eu, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁴Ir, ¹⁹⁷Hg, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At,²¹²Pb, ²¹²Bi and ²¹³Bi. Preferred therapeutic radionuclides include¹⁸⁸Re, ¹⁸⁶Re, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I,⁷⁷Br, ²¹¹At, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁹⁸Au, ¹⁶⁶Ho or ¹⁷⁷Lu. Conditions under whicha chelator will coordinate a metal are described, for example, by Gansowet al., U.S. Pat. Nos. 4,831,175, 4,454,106 and 4,472,509.

^(99m)Tc is a particularly attractive radioisotope for therapeutic anddiagnostic applications, as it is readily available to all nuclearmedicine departments, is inexpensive, gives minimal patient radiationdoses, and has ideal nuclear imaging properties. It has a half-life ofsix hours which means that rapid targeting of a technetium-labeledantibody is desirable. Accordingly, in certain preferred embodiments,the modified PSMA ligand includes a chelating agents for technium.

R can also be a radiosensitizing agents, e.g., a moiety that increasethe sensitivity of cells to radiation. Examples of radiosensitizingagents include nitroimidazoles, metronidazole and misonidazole (see:DeVita, V. T. Jr. in Harrison's Principles of Internal Medicine, p. 68,McGraw-Hill Book Co., N.Y. 1983, which is incorporated herein byreference). The modified PSMA ligand that comprises a radiosensitizingagent as the active moiety is administered and localizes at themetastasized cell. Upon exposure of the individual to radiation, theradiosensitizing agent is “excited” and causes the death of the cell.

There are a wide range of moieties which can serve as chelating ligandsand which can be derivatized to the PSMA inhibitors. For instance, thechelating ligand can be a derivative of1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA) and1-p-Isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid(ITC-MX). These chelators typically have groups on the side chain bywhich the chelator can be used for attachment to a PSMA inhibitor. Suchgroups include, e.g., benzylisothiocyanate, by which the DOTA, DTPA orEDTA can be coupled to, e.g., an amine group of the inhibitor.

In one embodiment, R is an “N_(x)S_(y)” chelate moiety. As definedherein, the term “N_(x)S_(y) chelates” includes bifunctional chelatorsthat are capable of coordinately binding a metal or radiometal and,preferably, have N₂S₂ or N₃S cores. Exemplary N_(x)S_(y) chelates aredescribed, e.g., in Fritzberg et al. (1988) PNAS 85:4024-29; and Weberet al. (1990) Bioconjugate Chem. 1:431-37; and in the references citedtherein.

The Jacobsen et al. PCT application WO 98/12156 provides methods andcompositions, i.e. synthetic libraries of binding moieties, foridentifying compounds which bind to a metal atom. The approach describedin that publication can be used to identify binding moieties which cansubsequently be added to PSMA ligands to derive the modified PSMAligands of the present invention.

A problem frequently encountered with the use of conjugated proteins inradiotherapeutic and radiodiagnostic applications is a potentiallydangerous accumulation of the radiolabeled moiety fragments in thekidney. When the conjugate is formed using a acid- or base-labilelinker, cleavage of the radioactive chelate from the protein canadvantageously occur. If the chelate is of relatively low molecularweight, as most of the subject modified PSMA ligands are expected to be,it is not retained in the kidney and is excreted in the urine, therebyreducing the exposure of the kidney to radioactivity. However, incertain instances, it may be advantageous to utilize acid- orbase-labile linkers in the subject ligands for the same reasons theyhave been used in labeled proteins.

Accordingly, certain of the subject modified PSMA ligands can besynthesized, by standard methods known in the art, to provide reactivefunctional groups which can form acid-labile linkages with, e.g., acarbonyl group of the ligand. Examples of suitable acid-labile linkagesinclude hydrazone and thiosemicarbazone functions. These are formed byreacting the oxidized carbohydrate with chelates bearing hydrazide,thiosemicarbazide, and thiocarbazide functions, respectively.

Alternatively, base-cleavable linkers, which have been used for theenhanced clearance of the radiolabel from the kidneys, can be used. See,for example, Weber et al. 1990 Bioconjug. Chem. 1:431. The coupling of abifunctional chelate to a PSMA ligand via a hydrazide linkage canincorporate base-sensitive ester moieties in a linker spacer arm. Suchan ester-containing linker unit is exemplified by ethyleneglycolbis(succinimidyl succinate), (EGS, available from Pierce ChemicalCo., Rockford, Ill.), which has two terminal N-hydroxysuccinimide (NHS)ester derivatives of two 1,4-dibutyric acid units, each of which arelinked to a single ethylene glycol moiety by two alkyl esters. One NHSester may be replaced with a suitable amine-containing BFC (for example2-aminobenzyl DTPA), while the other NHS ester is reacted with alimiting amount of hydrazine. The resulting hyrazide is used forcoupling to the PSMA ligand, forming an ligand-BFC linkage containingtwo alkyl ester functions. Such a conjugate is stable at physiologicalpH, but readily cleaved at basic pH.

PSMA ligands labeled by chelation are subject to radiation-inducedscission of the chelator and to loss of radioisotope by dissociation ofthe coordination complex. In some instances, metal dissociated from thecomplex can be re-complexed, providing more rapid clearance ofnon-specifically localized isotope and therefore less toxicity tonon-target tissues. For example, chelator compounds such as EDTA or DTPAcan be infused into patients to provide a pool of chelator to bindreleased radiometal and facilitate excretion of free radioisotope in theurine.

In still other embodiments, R is a Boron addend, such as a carborane.For example, carboranes can be prepared with carboxyl functions onpendant side chains, as is well known in the art. Attachment of suchcarboranes to an amine functionality, e.g., as may be provided on thePSMA ligand, can be achieved by activation of the carboxyl groups of thecarboranes and condensation with the amine group to produce theconjugate. Such modified PSMA ligands can be used for neutron capturetherapy.

In still other embodiments, the modified PSMA ligand includes acytotoxic moiety as the R functionality, such as a chemotherapeuticagent or a toxin. Many drugs and toxins are known which have cytotoxiceffects on cells, and can be used in connection with the presentinvention. They are to be found in compendia of drugs and toxins, suchas the Merck Index, Goodman and Gilman, and the like, and in thereferences cited above.

Chemotherapeutics useful as active moieties which when conjugated to amodified PSMA ligand are specifically delivered to metastasizedcolorectal cells are typically, small chemical entities produced bychemical synthesis. Chemotherapeutics include cytotoxic and cytostaticdrugs. Chemotherapeutics may include those which have other effects oncells such as reversal of the transformed state to a differentiatedstate or those which inhibit cell replication. Examples of knowncytotoxic agents useful in the present invention are listed, forexample, in Goodman et al., “The Pharmacological Basis of Therapeutics,”Sixth Edition, A. G. Gilman et al, eds./Macmillan Publishing Co. NewYork, 1980. These include taxol, nitrogen mustards, such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard andchlorambucil; ethylenimine derivatives, such as thiotepa; alkylsulfonates, such as busulfan; nitrosoureas, such as carmustine,lomustine, semustine and streptozocin; triazenes, such as dacarbazine;folic acid analogs, such as methotrexate; pyrimidine analogs, such asfluorouracil, cytarabine and azaribine; purine analogs, such asmercaptopurine and thioguanine; vinca alkaloids, such as vinblastine andvincristine; antibiotics, such as dactinomycin, daunorubicin,doxorubicin, bleomycin, mithramycin and mitomycin; enzymes, such asL-asparaginase; platinum coordination complexes, such as cisplatin;substituted urea, such as hydroxyurea; methyl hydrazine derivatives,such as procarbazine; adrenocortical suppressants, such as mitotane;hormones and antagonists, such as adrenocortisteroids (prednisone),progestins (hydroxyprogesterone caproate, medroprogesterone acetate andmegestrol acetate), estrogens (diethylstilbestrol and ethinylestradiol), antiestrogens (tamoxifen), and androgens (testosteronepropionate and fluoxymesterone).

Drugs that interfere with intracellular protein synthesis can also beused; such drugs are known to these skilled in the art and includepuromycin, cycloheximide, and ribonuclease.

Prodrugs forms of the chemotherapeutic moiety are especially useful inthe present invention to generate an inactive precursor.

Most of the chemotherapeutic agents currently in use in treating cancerpossess functional groups that are amenable to chemical crosslinkingdirectly with an amine or carboxyl group of a PSMA ligand. For example,free amino groups are available on methotrexate, doxorubicin,daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin andbleomycin while free carboxylic acid groups are available onmethotrexate, melphalan, and chlorambucil. These functional groups, thatis free amino and carboxylic acids, are targets for a variety ofhomobifunctional and heterobifunctional chemical crosslinking agentswhich can crosslink these drugs directly to a free amino group of a PSMAligand.

Peptide and polypeptide toxins are also useful as active moieties, andthe present invention specifically contemplates embodiments wherein R isa toxin. Toxins are generally complex toxic products of variousorganisms including bacteria, plants, etc. Examples of toxins includebut are not limited to: ricin, ricin A chain (ricin toxin), Pseudomonasexotoxin (PE), diphtheria toxin (DT), Clostridium perfringensphospholipase C (PLC), bovine pancreatic ribonuclease (BPR), pokeweedantiviral protein (PAP), abrin, abrin A chain (abrin toxin), cobra venomfactor (CVF), gelonin (GEL), saporin (SAP), modeccin, viscumin andvolkensin.

In addition, there are other active agents which can be used to create amodified PSMA ligand for the treatment of cancer. For example, modifiedPSMA ligands can be generated to include active enzyme. The modifiedPSMA ligand specifically localizes the activity to the tumor cells. Aninactive prodrug which can be converted by the enzyme into an activedrug is administered to the patient. The prodrug is only converted to anactive drug by the enzyme which is localized to the tumor. An example ofan enzyme/prodrug pair includes alkaline phosphatase/etoposidephosphate.In such a case, the alkaline phosphatase is conjugated to a PSMA ligand.The modified PSMA ligand is administered and localizes at themetastasized cell. Upon contact with etoposidephosphate (the prodrug),the etoposidephosphate is converted to etoposide, a chemotherapeuticdrug which is taken up by the cancer cell.

The present invention also contemplates dyes used, for example, inphotodynamic therapy, and used in conjunction with appropriatenon-ionizing radiation. The use of light and porphyrins in methods ofthe present invention is also contemplated and their use in cancertherapy has been reviewed. van den Bergh, Chemistry in Britain, 22:430-437 (1986), which is incorporated herein in its entirety byreference.

The modified PSMA ligands of the invention can be, for example,formulated as a solution, suspension or emulsion in association with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes may also be used. The vehicle may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously. The pharmaceutical compositions of thepresent invention may be administered by any means that enables themodified PSMA ligand to reach the targeted cells. In some embodiments,routes of administration include those selected from the groupconsisting of intravenous, intraarterial, intraperitoneal, localadministration into the blood supply of the organ in which the tumorresides or directly into the tumor itself. Intravenous administration isthe preferred mode of administration. It may be accomplished with theaid of an infusion pump.

The dosage administered varies depending upon factors such as: thenature of the active moiety; the nature of the modified PSMA ligand;pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment.

Because the subject ligands are specifically targeted to cells withPSMA/NAALADase activity, those modified PSMA ligands which comprisechemotherapeutics or toxins are administered in doses less than thosewhich are used when the chemotherapeutics or toxins are administered asunconjugated active agents, preferably in doses that contain up to 100times less active agent. In some embodiments, modified PSMA ligandswhich comprise chemotherapeutics or toxins are administered in dosesthat contain 10-100 times less active agent as an active moiety than thedosage of chemotherapeutics or toxins administered as unconjugatedactive agents. To determine the appropriate dose, the amount of compoundis preferably measured in moles instead of by weight. In that way, thevariable weight of different modified PSMA ligands does not affect thecalculation. Presuming a one to one ratio of modified PSMA ligand toactive moiety in modified PSMA ligands of the invention, less moles ofmodified PSMA ligands may be administered as compared to the moles ofunmodified PSMA ligands administered, preferably up to 100 times lessmoles.

(iv) Exemplary Uses of the Subject Ligands

A. PSMA Ligands in Treatment of Disease Conditions

The present invention also relates to a method of treating a prostatedisease in an animal, comprising administering an effective amount of acompound of formula I, II, III, IV or V to said animal. In a preferredembodiment, said prostate disease is prostate cancer such as prostaticadenocarcinoma, benign prostatic hyperplasia, or conditions involvingthe prostate requiring administration of the compounds of the presentinvention, such prostatic intraepithelial neoplasia (PIN).

In addition to prostate cancer, other forms of cancer that may betreated with the compounds of the present invention include withoutlimitation: ACTH-producing tumors, acute lymphocytic leukemia, acutenonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer,brain cancer, breast cancer, cervix cancer, chronic lymphocyticleukemia, chronic myelocytic leukemia, colorectal cancer, cutaneousT-cell lymphoma, endometrial cancer, esophageal cancer, Ewing's sarcoma,gallbladder cancer, hairy cell leukemia, head & neck cancer, Hodgkin'slymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer(small and/or non-small cell), malignant peritoneal effusion, malignantpleural effusion, melanoma, mesothelioma, multiple myeloma,neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovary cancer, ovary(germ cell) cancer, pancreatic cancer, penis cancer, retinoblastoma,skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomachcancer, testicular cancer, thyroid cancer, trophoblastic neoplasms,cancer of the uterus, vaginal cancer, cancer of the vulva and Wilm'stumor.

The compounds of the present invention are particularly useful intreating cancer of tissues where PSMA/NAALADase enzymes reside. Suchtissues include the prostate as well as the brain, kidney and testis.

For patients who initially present without advanced or metastaticcancer, the subject PSMA ligand-based drugs are used as an immediateinitial therapy prior to surgery and radiation therapy, and as acontinuous post-treatment therapy in patients at risk for recurrence ormetastasis (based upon high PSMA, high Gleason's score, locallyextensive disease, and/or pathological evidence of tumor invasion in thesurgical specimen). The goal in these patients is to inhibit the growthof potentially metastatic cells from the primary tumor during surgery orradiotherapy and inhibit the growth of tumor cells from undetectableresidual primary tumor.

For patients who initially present with advanced or metastatic cancer,PSMA ligand-based drugs are used as a continuous supplement to, orpossible as a replacement for hormonal ablation. The goal in thesepatients is to slow tumor cell growth from both the untreated primarytumor and from the existing metastatic lesions.

In addition, the invention may be particularly efficacious duringpost-surgical recovery, where the present compositions and methods maybe particularly effective in lessening the chances of recurrence of atumor engendered by shed cells that cannot be removed by surgicalintervention.

The present invention also includes a diagnostic kit for performing themethods of the present invention and may contain compounds and/orcompositions containing the compounds of the present invention. Forinstance, radiolabeled ligands may be used in a manner so as to providediagnostic information. Examples of diagnostic information and usesinclude determining the type of disease, the progress of the particulardisease, the location of cells targeted by the modified PSMA ligand andsimilar diagnostic uses known to persons skilled in the art.

In the methods of the present invention, the compounds may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir indosage formulations containing conventional non-toxicpharmaceutically-acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes subcutaneous, intravenous,intramuscular, intraperitoneal, intrathecal, intraventricular,intrasternal or intracranial injection and infusion techniques. Invasivetechniques are preferred, particularly direct administration to damagedneuronal tissue.

To be effective therapeutically as central nervous system targets, thecompounds of the present invention should readily penetrate theblood-brain barrier when peripherally administered. Compounds whichcannot penetrate the blood-brain barrier can be effectively administeredby an intraventricular route.

The compounds may also be administered in the form of sterile injectablepreparations, for example, as sterile injectable aqueous or oleaginoussuspensions. These suspensions can be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparations may also besterile injectable solutions or suspensions in non-toxicparenterally-acceptable diluents or solvents, for example, as solutionsin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile fixed oils are conventionally employed assolvents or suspending mediums. For this purpose, any bland fixed oilsuch as a synthetic mono- or di-glyceride may be employed. Fatty acidssuch as oleic acid and its glyceride derivatives, including olive oiland castor oil, especially in their polyoxyethylated forms, are usefulin the preparation of injectables. These oil solutions or suspensionsmay also contain long-chain alcohol diluents or dispersants.

Additionally, the compounds may be administered orally in the form ofcapsules, tablets, aqueous suspensions or solutions. Tablets may containcarriers such as lactose and corn starch, and/or lubricating agents suchas magnesium stearate. Capsules may contain diluents including lactoseand dried corn starch. Aqueous suspensions may contain emulsifying andsuspending agents combined with the active ingredient. The oral dosageforms may further contain sweetening and/or flavoring and/or coloringagents.

The compounds may further be administered rectally in the form ofsuppositories. These compositions can be prepared by mixing the drugwith suitable non-irritating excipients which are solid at roomtemperature, but liquid at rectal temperature such that they will meltin the rectum to release the drug. Such excipients include cocoa butter,beeswax and polyethylene glycols.

Moreover, the compounds may be administered topically, especially whenthe conditions addressed for treatment involve areas or organs readilyaccessible by topical application, including neurological disorders ofthe eye, the skin or the lower intestinal tract.

For topical application to the eye, or ophthalmic use, the compounds canbe formulated as micronized suspensions in isotonic, pH adjusted sterilesaline or, preferably, as a solution in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, the compounds may be formulated into ointments,such as petrolatum.

For topical application to the skin, the compounds can be formulatedinto suitable ointments containing the compounds suspended or dissolvedin, for example, mixtures with one or more of the following: mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the compounds can be formulated into suitable lotions orcreams containing the active compound suspended or dissolved in, forexample, a mixture of one or more of the following: mineral oil,sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

Topical application to the lower intestinal tract can be effected inrectal suppository formulations (see above) or in suitable enemaformulations.

The compounds of the present invention may be administered by a singledose, multiple discrete doses or continuous infusion. Since thecompounds are small, easily diffusible and relatively stable, they arewell suited to continuous infusion. Pump means, particularlysubcutaneous pump means, are preferred for continuous infusion.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, modified PSMA ligands may beincorporated into a polymer matrix for controlled release over a periodof days. Such controlled release films are well known to the art.Examples of polymers commonly employed for this purpose that may be usedin the present invention include nondegradable ethylene-vinyl acetatecopolymer and degradable lactic acid-glycolic acid copolymers. Certainhydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol)also may be useful.

Dose levels on the order of about 0.1 mg to about 10,000 mg of theactive ingredient compound are useful in the treatment of the aboveconditions, with preferred levels being about 0.1 mg to about 1,000 mg.The specific dose level for any particular patient will vary dependingupon a variety of factors, including the activity of the specificcompound employed; the age, body weight, general health, sex and diet ofthe patient; the time of administration; the rate of excretion; drugcombination; the severity of the particular disease being treated; andthe form of administration. Typically, in vitro dosage-effect resultsprovide useful guidance on the proper doses for patient administration.Studies in animal models are also helpful, particularly in determiningeffective doses for treating cancer. The considerations for determiningthe proper dose levels are well known in the art.

For the methods of the present invention, any administration regimenregulating the timing and sequence of drug delivery can be used andrepeated as necessary to effect treatment. Such regimen may includepretreatment and/or co-administration with additional therapeuticagents.

For patients with prostate cancer that is neither advanced normetastatic, the compounds of the present invention may be administered(i) prior to surgery or radiation treatment to reduce the risk ofmetastasis; (ii) during surgery or in conjunction with radiationtreatment; and/or (iii) after surgery or radiation therapy to reduce therisk of recurrence and to inhibit the growth of any residual tumorouscells.

For patients with advanced or metastatic prostate cancer, the compoundsof the present invention may be administered as a continuous supplementto, or as a replacement for, hormonal ablation in order to slow tumorcell growth in both the untreated primary tumor and the existingmetastatic lesions.

The methods of the present invention are particularly useful where shedcells could not be removed by surgical intervention. After post-surgicalrecovery, the methods of the present invention would be effective inreducing the chances of recurrence of a tumor engendered by such shedcells.

The modified PSMA ligands of the instant invention can also be used todetermine the abundance of PSMA in a sample, based on their ability tobind PSMA. For example, the sample containing PSMA may be incubated withone of the modified PSMA ligands of the instant invention to allowbinding between PSMA and its modified ligand. Bound ligands can then beseparated from unbound ones, and the abundance of the bound PSMA ligandscan be determined conveniently, especially when the abundance of themodifying group (the “R Group”) is to be measured. The amount of boundmodified PSMA ligands is partly determined by the binding affinitybetween the ligand and PSMA, it is also determined by the amount of PSMAin the sample. The relative amount of PSMA within different samples canbe compared directly. However, when an absolute amount of PSMA within asample is desired, a series of control samples with known PSMAconcentrations can be employed to derive a standard curve, from whichthe absolute concentration of PSMA can be deduced.

There are many ways to determine the abundance of bound PSMA ligands.For example, if the modifying group is a fluorescent tag, the amount offluorescent signals is a measurement of the ligand abundance. Similarly,the amount of chelate groups can be determined by the amount ofradio-isotopes that can be bound by the chelate groups. Otherembodiments of the invention will be apparent to skilled artisans.

Separation of bound and unbound PSMA ligands can be achieved in manyconceivable ways. For example, PSMA within a test sample can be fixed ona solid support, such as in a well of a microtiter plate. Excessiveunbound PSMA ligands, just like in an ELISA assay, can then be washedaway. Alternatively, soluble PSMA can be immunoprecipitated using aPSMA-specific antibody, and the amount of the associated PSMA ligandsdetermined using the methods described before.

B. Combination with Other Treatments

(i) Surgery and Radiation Treatment

In general, surgery and radiation treatment are employed as potentiallycurative therapies for patients with localized prostate cancer who areunder 70 years of age and are expected to live at least 10 more years.

Approximately 70% of newly diagnosed prostate cancer patients fall intothis category. Approximately 90% of these patients (65% of totalpatients) undergo surgery, while approximately 10% of these patients (7%of total patients) undergo radiation treatment.

Histopathological examination of surgical specimens reveals thatapproximately 63% of patients undergoing surgery (40% of total patients)have locally extensive tumors or regional (lymph node) metastasis thatwas undetected at initial diagnosis. These patients are at asignificantly greater risk of recurrence. Approximately 40% of thesepatients will actually develop recurrence within five years aftersurgery. Results after radiation treatment are even less encouraging.Approximately 80% of patients who have undergone radiation treatment astheir primary therapy have disease persistence or develop recurrence ormetastasis within five years after treatment.

Currently, most prostate cancer patients undergoing surgery andradiation treatment do not receive any immediate follow-up therapy.Rather, they are monitored frequently for elevated PSMA, which is theprimary indicator of recurrence or metastasis.

Based on the above statistics, there is considerable opportunity to usethe present invention in conjunction with surgery and/or radiationtreatment.

(ii) Hormonal Therapy

Hormonal ablation is the most effective palliative treatment for the 10%of patients with metastatic prostate cancer. Hormonal ablation bymedication and/or orchiectomy is used to block hormones that promotefurther growth and metastasis of prostate cancer. With time, both theprimary and metastatic tumors of virtually all of these patients becomehormone-independent and resistant to therapy. Approximately 50% ofpatients with metastatic cancer die within three years after initialdiagnosis, and 75% of such patients die within five years afterdiagnosis. Continuous supplementation with the compounds of the presentinvention may be used to prevent or reverse this potentiallymetastasis-permissive state.

(iii) Chemotherapy

While chemotherapy has been successful in treating some forms of cancer,it has shown slight therapeutic value in treating prostate cancer whereit is generally reserved as a last resort. Accordingly, the opportunityto treat prostate cancer by combining chemotherapy with the methods ofthe present invention will be rare. When combined, however, suchtreatments should be more effective than chemotherapy alone incontrolling prostate cancer.

(iv) Immunotherapy

The compounds of the present invention may also be used in combinationwith monoclonal antibodies to treat prostate cancer. Such combinedtreatment is particularly effective for patients with pelvic lymph nodeinvolvement, of which only 34% survive after 5 years. An example of suchmonoclonal antibodies is cell membrane-specific anti-prostate antibody.

The present invention may also be used with immunotherapies based onpolyclonal or monoclonal antibody-derived reagents. Monoclonalantibody-derived reagents are preferred. These reagents are well knownin the art, and include radiolabeled monoclonal antibodies such asmonoclonal antibodies conjugated with strontium-89.

(v) Cryotherapy

The methods of the present invention may also be used in conjunctionwith cryotherapy for treatment of prostate cancer.

(vi) Chemical Prostatectomy

The methods and compositions of the invention may also be used forchemical ablation of the prostate gland, or “chemical prostatectomy.”Chemical prostatectomy may be used as a non-surgical means of treatmentfor an individual with a diseased prostate. Alternatively, such a methodmay be used as a preventive treatment for individuals at high risk ofdeveloping a prostate associated disease or disorder, e.g., individualswith a positive family history or personal history of prostate disease.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Conjugation of Guilford 11254-36 to the Near-InfraredFluorophore IRDye78

To demonstrate that PSMA ligands can be functionalized according to theinstant invention, compound Guilford 11254-36 was conjugated underaqueous conditions to the near-infrared fluorophore IRDye78 (LI-COR,Lincoln, Nebr.; excitation 771 nm, emission 796 nm) using the chemistryshown in FIG. 1A. IRDye78 is a near-infrared fluorophore that can beused in the intraoperative detection of cancer cells in a surgicalfield.

The desired product of the reaction could be easily separated fromreactants using normal phase thin-layer chromatography (FIG. 1B). Askilled artisan would be able to utilize other commonly known techniquesto isolate, or purify the conjugated chemical product.

EXAMPLE 2 Near-Infrared Fluorescent Detection of PSMA in Living Cellsusing a Functionalized PSMA Ligand

To demonstrate that the compound of the instant invention(functionalized PSMA ligand) still retains the ability to bind PSMA,just like the unconjugated PSMA ligand, the compound Guilford 11254-36was conjugated to the near-infrared fluorophore IRDye78 as describedabove. COS-7 cells, which normally do not express PSMA, were infectedwith an adenovirus that either co-express GFP and PSMA (left column), orexpresses GFP only (right column). 72 hours after infection, cells wereincubated with 5 μM CaCl₂ for 15 minutes at 37° C. Cells were thenwashed 3× with PBS supplemented with 10 μM MgCl₂ and 1 μM CaCl₂ andviewed under a fluorescent microscope using various filter sets.

FIG. 2 middle panels shows that COS-7 cells were infected efficiently bythe adenovirus and many cells in the view field expressed the GFP marker(and presumably also the PSMA protein in the left column). Althoughthere is no morphological differences between the GFP expressing andGFP-PSMA co-expression cells (FIG. 2, bottom panels), it is clear thatthe functionalized PSMA ligand, IRDye78 conjugated to Guilford 11254-36,can bind GFP-PSMA co-expressing cells (FIG. 2, top panel, left column),as is evident from the near-infrared fluorescence (excitation 771 nm,emission 796 nm). The strong plasma membrane Guilford/IRDye78 signaldemonstrate that the PSMA protein is correctly localized the its usualplasma membrane localization. In contrary, control GFP-expressing cellsdo not bind Guilford/IRD78 conjugate (FIG. 2, top panel, right column).

EXAMPLE 3 Proof of Specificity of the Modified PSMA Ligand

To demonstrate that the functionalized PSMA ligand retains the bindingspecificity to PSMA, COS-7 cells were infected with an adenovirus thateither co-express GFP and PSMA (left column), or co-expresses GFP andErb-B2 (a breast cancer protein; right column). 72 hours aftertransfection, cells were incubated with 5 μM of the Guilford/IRDye78conjugate in PBS supplemented with 10 μM MgCl₂ and 1 μM CaCl₂ for 15minutes at 37° C. Cells were then washed 3× with PBS supplemented with10 μM CaCl₂, fixed in 2% paraformaldehyde for 10 minutes at roomtemperature, washed, and viewed under a fluorescent microscope usingvarious filter sets.

In FIG. 3, top panel shows near-infrared fluorescence (excitation 771nm, emission 796 nm). Middle panel shows green fluorescent proteinsignal. Bottom panel shows phase contrast of same field. Arrow points torepresentative cell with strong plasma membrane Guilford/IRDye78 signalin cells making PSMA. No binding is seen with cells expressing Erb-B2(right panel). This demonstrates that the functionalized PSMA ligandretains the binding specificity of the unconjugated PSMA ligand.

Similar experiments have also been performed in a second cell-type, PC-3cells, a human prostate cancer cell line which normally do not expressPSMA. Essentially the same results were obtained as described above forCOS-7 cells (FIG. 4).

EXAMPLE 4 PSMA Ligands and IRDye78 Inhibit PSMA Activity

To determine the effects of several PSMA ligands and modified PSMAligands (β-AG/78) on PSMA activity, ³H-NAAG assay (Slusher et al., J.Biol. Chem., 1990, 265: 21297-301) was used to measure the activity ofPSMA at the presence or absence of several PSMA ligands.

β-AG/78 (FIG. 5, bottom panel) is a conjugate of β-AG and thenear-infrared fluorophore IRDye78. The conjugate can be synthesizedusing a similar scheme as shown in Example 1, and the product can bepurified using either TLC (not shown) or HPLC, as shown in FIG. 6. As acontrol, IRDye78 was also tested. The results were shown as percentageof remaining PSMA activity as compared to PSMA activity without anyadditives, over a range of inhibitor concentrations.

The result shown in FIG. 5, top panel, demonstrated that β-AG is indeedan inhibitor of PSMA enzymatic activity as measured by the ³H-NAAGassay. The inhibitory activity of β-AG was slightly less potent thanthat of beta-N-acetyl-AG (β-NAAG). Surprisingly, the IRDye78 is also aninhibitor of PSMA. In addition, the inhibitory effect of IRDye78 andβ-AG are synergistic so that the β-AG/78 conjugate is a much more potentinhibitor than either β-AG or IRDye78 alone.

Similar results were obtained from a different set of experiments, wherea Guilford 11254-36/IRDye78 conjugate (synthesis see Example 1) wasfound to be a more potent inhibitor than either Guilford 11254-36 (FIG.7, bottom panel) or IRDye78 alone (FIG. 7, top panel), indicating thatthe inhibitory effects of IRDye78 and the Guilford compound aresynergistic.

EXAMPLE 5 Modified PSMA Ligands Bind Directly to PSMA

As shown in the fluorescence polarization data of FIG. 8, bothbeta-AG/78 and Guilfrod/78 binds directly to PSMA with a K_(d)essentially equivalent to their respective IC₅₀'s of enzyme inhibition.This means that IC₅₀ can be used as a surrogate for affinity, and alsopredicts that the conjugates presented herein can be used asvisualization agents for PSMA-positive cells. Indeed, this is shown tobe the case in experiments conducted in both COS-7 cells and PC-3 cells(see Examples 2 and 3).

EXAMPLE 6 Serum Stability of the Modified PSMA Ligand

It was determined that Guilford/78 is stable for up to 1 hour whenincubated at 37° C. in human serum (not shown). We are presently testingdetection in vivo in mouse xenograft models of prostate cancer, usingthe conjugated PSMA ligands.

Equivalents

It should be understood that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The references cited hereinabove are all incorporated herein byreference.

1-3. (canceled)
 4. A compound represented in the general formula (Ie):

wherein: X represents O or S; Y represents:

R represents a chelate ligand, a fluorescence tag, or a cytotoxicmoiety; R1 and R3, independently for each occurrence, represents analkyl, an alkenyl, a cycloalkyl, a cycloalkenyl, an aryl,—(CH₂)_(m)-aryl, -alkyl-CO₂R4, -alkenyl-CO₂R4, -cycloalkyl-CO₂R4,-cycloalkenyl-CO₂R4 or -aryl-CO₂R4; R2 and R4, independently for eachoccurrence, represent hydrogen, a lower alkyl, or a pharmaceuticallyacceptable salt; D₁ represents O or S; D₂ represents N₃, SH₂, NH₂, orNO₂; m is 1, 2, 3 or 4; and, n is 0, 1, 2 or
 3. 5-66. (canceled)
 67. Acompound represented by the formula VI, or a pharmaceutically acceptablesalt thereof:

wherein R represents a chelate ligand, a fluorescence tag, or acytotoxic moiety, and n is 0, 1, 2 or
 3. 68. The compound of claim 67,wherein the fluorescence tag is IRDye78, and the compound is representedby the formula VI(a):


69. The compound of claim 67, wherein the chelate ligand is aradioscintigraphic probe.
 70. The compound of claim 69, wherein thecompound is represented by the formula VI(b):


71. The compound of claim 67, wherein the cytotoxic moiety is acytotoxic drug for prostate cancer treatment.
 72. The compound of claim71, wherein the compound is represented by the formula VI(c):


73. A pharmaceutical composition comprising the compound or salt thereofof claim 67, and a pharmaceutically acceptable carrier.
 74. Thepharmaceutical composition of claim 67, wherein the fluorescence tag isIRDye78, and the compound is represented by the formula VI(a).
 75. Thepharmaceutical composition of claim 67, wherein the chelate ligand is aradioscintigraphic probe.
 76. The pharmaceutical composition of claim67, wherein the chelate ligand is a radioscintigraphic probe, and thecompound is represented by the formula VI(b).
 77. The pharmaceuticalcomposition of claim 68, wherein the cytotoxic moiety is a cytotoxicdrug for prostate cancer treatment.
 78. The pharmaceutical compositionof claim 68, wherein the cytotoxic moiety is a cytotoxic drug forprostate cancer treatment, and the compound is represented by theformula VI(c).
 79. A method to treat a patient suffering from a diseasecondition associated with PSMA-overexpression, comprising administeringto the patient an effective amount of the compound or salt of claim 67,or pharmaceutical composition thereof.
 80. The method of claim 79,wherein the disease condition is prostatic hyperplasia or prostatecancer.
 81. The method of claim 79, wherein the chelate ligand is aradioscintigraphic probe, and the compound is represented by the formulaVI(b).
 82. The method of claim 79, wherein the cytotoxic moiety is acytotoxic drug for prostate cancer treatment, and the compound isrepresented by the formula VI(c).